The present invention relates to viral vaccines. In particular, it relates to genetically engineered mutant viruses for use as vaccines; vaccines comprising the mutant viruses; recombinant cell; and to methods relating to the production of vaccines.
Viral vaccines are traditionally of two sorts. The first sort are xe2x80x98killedxe2x80x99 vaccines, which are virus preparations which have been killed by treatment with a suitable chemical such as beta-propiolactone. The second type are live xe2x80x98attenuatedxe2x80x99 vaccines, which are viruses which have been rendered less pathogenic to the host, either by specific genetic manipulation of the virus genome, or, more usually, by passage in some type of tissue culture system. These two types of vaccine each have their own disadvantages. Since killed vaccines do not replicate in the host, they must be administered by injection, and hence may generate an inappropriate kind of immune response. For example the Salk vaccine, a killed preparation of poliovirus, produces an immunoglobulin (Ig) G antibody response, but does not stimulate the production of IgA in the gut, the natural site of primary infection. Hence this vaccine, though it can protect the individual from the neurological complications of poliomyelitis, does not block primary infection, and so does not confer xe2x80x9cherd immunityxe2x80x9d. In addition, killed viruses, do not enter and replicate inside host cells. Hence any beneficial immunological response to non-structural proteins produced during replication is not available. They also cannot stimulate the production of cytotoxic T cells directed against virus antigens. xe2x80x9cDeadxe2x80x9d antigens can be picked up by antigen presenting cells and presented to T cells. However, the presentation occurs via MHC Class II molecules and leads to stimulation of T helper cells. In turn, the T helper cells help B cells to produce specific antibody against the antigen. In order to stimulate the production of cytotoxic T cells, virus antigens must be processed through a particular pathway inside the infected cell, and presented as broken-up peptide fragments on MHC Class I molecules. This degradation pathway is thought to work most effectively for proteins that are synthesised inside the infected cell, and hence only virus that enters host cells and expresses immunogenic viral protein is capable of generating virus-specific cytotoxic T cells.Therefore, killed vaccines are poor inducers of cellular immunity against virus infection. From this point of view, live attenuated vaccines are more satisfactory.
To date, live attenuated viruses have been made by deleting an inessential gene or partly damaging one or more essential genes (in which case, the damage is such that the genes are still functional, but do not operate so effectively). However, live attenuated viruses often retain residual pathogenicity which can have a deleterious effect on the host. In addition, unless the attenuation is caused by a specific deletion, there remains the possibility of reversion to a more virulent form. Nevertheless, the fact that some viral protein production occurs in the host means that they are often more effective than killed vaccines which cannot produce such viral protein.
Live attenuated viruses, as well as being used as vaccines in their own right, can also be used as xe2x80x98vaccine vectorsxe2x80x99 for other genes, in other words carriers of genes from a second virus (or other pathogen) against which protection is required. Typically, members of the pox virus family eg. vaccinia virus, are used as vaccine vectors. When a virus, is used as a vaccine vector, it is important that it causes no pathogenic effects. In other words it may need to be attenuated in the same way that a simple virus vaccine is attenuated. The same disadvantages as those described above, therefore apply in this case.
It has been found possible to delete a gene from a viral genome and provide a so-called xe2x80x98complementingxe2x80x99 cell which provides the virus with the product of the deleted gene. This has been achieved for certain viruses, for example adenoviruses, herpesviruses and retroviruses. For adenoviruses, a human cell line was transformed with fragments of adenovirus type 5 DNA (Graham, Smiley, Russell and Nairn, J. Gen. Virol., 36,59-72, 1977). The cell line expressed certain viral genes, and it was found that it could support the growth of virus mutants which had those genes deleted or inactivated (Harrison, Graham and Williams, Virology 77, 319-329, 1977). Although the virus grew well on this cell line (the xe2x80x98complementing cell linexe2x80x99) and produced standard viral particles, it could not grow at all on normal human cells. Cells expressing the T-antigen-encoding region of the SV40 virus genome (a papovavirus) have also been shown capable of supporting the replication of viruses specifically deleted in this region (Gluzman, Cell, 23,182-195, 1981). For herpes simplex virus, cell lines expressing the gB glycoprotein (Cai et al, J. Virol. 62,714-721, 1987) the gD glycoprotein (Ligas and Johnson, J. Virol. 62,1486, 1988) and the Immediate Early protein ICP4 (Deluca et al., J. Virol., 56,558, 1985) have been produced, and these have been shown capable of supporting the replication of viruses with specifically inactivated copies of the corresponding genes.
The present invention provides a mutant virus for use as a vaccine, in which a viral gene encoding a protein which is essential for the production of infectious virus has been deleted or inactivated; and wherein said virus can be grown in a cell which has a heterologous nucleotide sequence which allows said cell to express the essential protein encoded by said deleted or inactivated viral gene.
The present invention also provides a vaccine which comprises a virus as described above, together with one or more excipients and/or adjuvants. The viral genome may itself provide the immunogen, or it may contain a heterologous gene insert expressing the immunogenic protein.
The present invention also provides a complementing cell transfected with an attenuated virus as described above, for use in the preparation of a vaccine.
The present invention also provides a method which comprises the use of a virus as described above in the preparation of a vaccine for the therapeutic or prophylactic treatment of a disease.
The present invention also provides a method for the production of a vaccine which comprises: culturing a cell infected with a virus having a deleted or inactivated viral gene encoding a protein which is essential for the production of infectious virus, and wherein the host cell has a heterologous nucleotide sequence comprising said viral gene and which is able to express the essential protein encoded by said gene; harvesting the virus thus produced, and using it in a vaccine.
The virus may be derived from herpes simplex virus (HSV) in which, for example, the gene encoding glycoprotein H (gH) has been inactivated or deleted. The mutant virus may also comprise a heterologous sequence encoding an immunogen derived from a pathogen. The host cell will suitably be a recombinant eukaryotic cell line containing the gene encoding HSV glycoprotein H. As another example the virus may be derived from an orthopox virus, for example, vaccinia virus, which again may comprise a heterologous sequence encoding an immunogen derived from a pathogen.
This invention shows a unique way of combining the efficacy and safety of a killed vaccine with the extra immunological response induced by the in vivo production of viral protein by the attenuated vaccine. In preferred embodiments it comprises two features. Firstly, a selected gene is inactivated within the virus genome, usually by creating a specific deletion. This gene will be involved in the production of infectious virus, but preferably not preventing replication of the viral genome. Thus the infected cell can produce more viral protein from the replicated genetic material, and in some cases new virus particles may be produced, but these would not be infectious. This means that the viral infection cannot spread from the site of inoculation.
A second feature of the invention is a cell which provides the virus with the product of the deleted gene, thus making it possible to grow the virus in tissue culture. Hence, although the virus lacks a gene encoding an essential protein, if it is grown in the appropriate host cell, it will multiply and produce complete virus particles which are to outward appearances indistinguishable from the original virus. This mutant virus preparation is inactive in the sense that it has a defective genome and cannot produce infectious virus in a normal host, and so may be administered safely in the quantity required to generate directly a humoral response in the host. Thus, the mutant virus need not be infectious for the cells of the host to be protected and merely operates in much the same way as a conventional killed or attenuated virus vaccine. However, preferably the immunising virus is itself still infectious, in the sense that it can bind to a cell, enter it, and initiate the viral replication cycle and is therefore capable of initiating an infection within a host cell of the species to be protected, and producing therein some virus antigen. There is thus the additional opportunity to stimulate the cellular arm of the host immune system.
The deleted or inactivated gene is preferably one involved as late as possible in the viral cycle, so as to provide as many viral proteins as possible in vivo for generating an immunogenic response. For example, the gene may be one involved in packaging or some other post-replicative event, such as the gH glycoprotein of HSV. However, the selected gene may be one involved in the viral genome replication, and the range of proteins expressed in vivo will depend upon the stage at which that gene is normally expressed. In the case of human cytomegalovirus (HCMV) the selected gene may be one (other than the Immediate Early gene) that effectively prevents viral genome replication in vivo, since the Immediate Early gene which is produced prior to viral genome replication (and indeed is essential for it) is highly immunogenic.
This invention can be applied to any virus where one or more essential gene(s) can be identified and deleted from or inactivated within the virus genome. For DNA viruses, such as Adeno, Herpes, Papova, Papilloma and Parvo viruses, this can be achieved directly by (i) the in vitro manipulation of cloned DNA copies of the selected essential gene to create specific DNA changes; and (ii) re-introduction of the altered version into the virus genome through standard procedures of recombination and marker rescue. The invention however, is also applicable to RNA viruses. Techniques are now available which allow complementary DNA copies of a RNA virus genome to be manipulated in vitro by standard genetic techniques, and then converted to RNA by in vitro transcription. The resulting RNAs may then be re-introduced into the virus genome. The technique has been used to create specific changes in the genome of both positive and negative stranded RNA viruses, e.g. poliovirus (Racaniello and Baltimore, Science, 214, 916-919, 1981) and influenza virus (Lutyes et al., Cell, 59, 1107-1113, 1989).
In theory, any gene encoding an essential protein should be a potential target for this approach to the creation of attenuated viruses. In practice however, the selection of the gene will be driven by a number of considerations.
1. The gene should preferably be one which is required later in infection. Thus replication of the attenuated virus is not interrupted in the early phase. This means that most and possibly all other virus antigens will be produced in the infected cell, and presented to the host immune system in conjunction with host cell MHC class 1 molecules. Such presentation leads to the development of cellular immunity against virus infection through the production of cytotoxic T cells. The cytotoxic T cells can recognise these antigens, and therefore kill virus infected cells. It is possible that the deleted gene could represent one which is not required at all for virus assembly, but is necessary for the assembled virus to be able to infect new cells. An example of such a protein is the HSV gH protein. In the absence of this protein, HSV virions are still produced, but they are non-infectious.
2. Ideally, the product of the selected gene should not, on its own, be toxic to the eukaryotic cell, so that a complementing cell can be produced relatively easily. This however is not an absolute requirement, since the gene may be placed under the control of an inducible promoter in the complementing cell, such that its expression may be switched on only when required.
The nature of the mutation created in the target gene is also a matter of choice. Any change which produces a non-functional gene product is satisfactory, as long as the risk of reversion to a wild type structure is minimised. Such changes include interruption of the target with extraneous sequences and creation of specific deletions. The most satisfactory strategy for a vaccine to be used as a therapeutic and/or prophylactic however, would be one where a deletion is made that encompasses the entire sequence to be introduced into the complementing cell. The approach minimises the risk of regenerating wild type virus through recombination between the virus and cell DNA in the complementing cell.
Although there are several examples of combinations of specifically inactivated viruses and complementing cells, (see earlier discussion), to date, these have been used either for basic research on the virus, or, as in the case of retroviruses, to make a safer vector for producing transgenic animals. They have not been used for vaccine purposes, and to the applicants knowledge no suggestion of this kind of use has been proposed.
As well as using such an inactivated virus/complementing cell combination to produce safe vaccines against the wild-type virus, this invention also deals with the use of the same system to produce safe viral vectors for use as vaccines against foreign pathogens.
An example of such a vector is one based on HSV. The HSV genome is large enough to accommodate considerable additional genetic information and several examples of recombinant HSV viruses carrying and expressing foreign genetic material have been described (e.g. Ligas and Johnson, J. Virol. 1988, op. cit.). Thus a virus with a deletion in an essential virus gene as described above, and also carrying and expressing a defined foreign gene, could be used as a safe vector for vaccination to generate an immune response against the foreign protein.
A particular characteristic of HSV is that it may become latent in neurones of infected individuals, and occasionally reactivate leading to a local lesion. Thus an HSV with a deletion in an essential virus gene and expressing a foreign gene could be used to produce deliberately latent infection of neurones in the treated individual. Reactivation of such a latent infection would not lead to the production of a lesion, since the virus vector would be unable to replicate fully, but would result in the onset of the initial part of the virus replication cycle. During this time expression of the foreign antigen could occur, leading to the generation of immune response. In a situation where the deleted HSV gene specified a protein which was not needed for virus assembly, but only for infectivity of assembled virions, such a foreign antigen might be incorporated into the assembled virus particles, leading to enhancement of its immunogenic effect. This expression of the foreign gene and incorporation of its protein in a viral particle could of course also occur at the stage where the mutant virus is first produced in its complementing host, in which case the mutant virus when used as a vaccine could present immediately the foreign protein to the species being treated.
In another example, vaccinia virus, a poxvirus, can carry and express genes from various pathogens, and it has been demonstrated that these form effective vaccines when used in animal experimental systems. The potential for use in humans is vast, but because of the known side effects associated with the widespread use of vaccinia as a vaccine against smallpox, there is reluctance to use an unmodified vaccinia virus on a large scale in humans. There have been attempts to attenuate vaccinia virus by deleting non-essential genes such as the vaccinia growth factor gene (Buller, Chakrabarti, Cooper, Twardzik and Moss, J. Virology 62,866-874, 1988). However, such attenuated viruses can still replicate in vivo, albeit at a reduced level. No vaccinia virus with a deletion in an essential gene has yet been produced, but such a virus, deleted in an essential gene as described above, with its complementing cell for growth, would provide a safer version of this vaccine vector.
A further advantage of this general strategy for immunisation against heterologous proteins is that it may be possible to perform multiple effective vaccinations with the same virus vector in a way not possible with conventional live virus vectors. Since a standard live virus vaccine probably relies for its efficacy on its ability to replicate in the host animal through many cycles of infection, its usefulness will be severely curtailed in an individual with immunity against that virus. Thus a second challenge with the same virus, whether to provide a booster immunisation against the same protein, or a new response against a different protein, is likely to be ineffective. Using a virus vector with a deletion in an essential gene however, where multi-cycle replication is not desired or required, the events leading to effective immunisation will occur very soon after immunisation. The dose of the mutant virus can be relatively large (since it should be completely safe), and it is therefore unlikely that these early events will be blocked by the host immune response, which will require some time to be mobilised completely.
Although we have referred above to a mutant virus being defective in an essential gene, and optionally containing a gene for an immunogenic pathogen protein, the mutant could be defective in more than one essential gene, and/or contain more than one immunogenic pathogen protein gene. Thus, the mutant virus might include the gene for HIV gp 120, to act as a vaccine in the manner suggested above, and also the gene for the HIV gag protein to be expressed within the vaccinated host and presented at the surface of the host cell in conjunction with MHC-I to stimulate a T-cell response in the host.